1. Field of the Invention
The present invention relates to a kit for and a method of amplifying a target nucleic acid using rolling circle amplification (RCA) method in the presence of a ligase and a restriction enzyme.
2. Description of the Related Art
In rolling circle amplification (RCA) method, a polymerization reaction is carried out at a temperature, for example, at room temperature, using a single-stranded (or “single-strand”) circular DNA as a template and a primer which is complementary to the single-strand circular DNA, to synthesize multiple continuous single-stranded linear copies of the template DNA. The RCA method has a high degree of sensitivity and thus has been successfully used for amplification of DNA from small amounts of starting material, but it is difficult to prepare a single-strand circular DNA template itself A single-strand circular DNA can be chemically synthesized or prepared through a separate reaction.
U.S. Pat. No. 5,714,320 discloses a method of synthesizing a selected oligonucleotide having well-defined ends, which include steps: (a) annealing an effective amount of an oligonucleotide primer to a single-strand circular template to yield a primed circular template, wherein the single-strand circular template includes (i) at least one copy of a nucleotide sequence complementary to the sequence of the selected oligonucleotide and (ii) at least one nucleotide effective to produce a cleavage site in the oligonucleotide multimer; (b) combining the primed circular template with an effective amount of at least two types of nucleotide triphosphates and an effective amount of a polymerase enzyme without the addition of auxiliary proteins to yield a single-strand oligonucleotide multimer complementary to the circular oligonucleotide template, wherein the oligonucleotide multimer includes multiple copies of the selected oligonucleotide; and (c) cleaving the oligonucleotide multimer at the cleavage site to produce the selected oligonucleotide having well-defined ends. Also, Proc. Natl. Acad. Sci. USA, Vol. 92, pp. 4641-4645 (May in 1995) discloses that RCA is used to amplify a DNA circle as short as 34 bp.
However, synthesis of the single-strand circular DNA template and amplification of a target nucleic acid have not been performed in the same vessel. In addition, a method of performing RCA in the presence of both a ligase and an endonuclease has not been disclosed.